Impact of hospital environmental cleaning with a potassium peroxymonosulphate-based environmental disinfectant and antimicrobial stewardship on the reduction of hospital-onset Clostridioides difficile infections.

BACKGROUND: A 1% potassium peroxymonosulphate-based environmental disinfectant (PPED) produces sodium hypochlorite when combined with sodium chloride, which functions as a disinfectant. However, little is known about the impact of hospital cleaning with PPED on hospital-onset Clostridioides difficile infection (HO-CDI). AIM: To reduce HO-CDI, we promoted antimicrobial stewardship and hospital ward cleaning with PPED: this study was conducted to evaluate their impact. METHODS: We began a promotion of post-prescription review with feedback for broad-spectrum antimicrobials and hospital ward cleaning with PPED. We reviewed the ratio of HO-CDI, PPED consumption, and days of therapy (DOT) of broad-spectrum antimicrobials between July 2014 and March 2018, dividing this time into the pre-promotion (July 2014 to June 2015) and post-promotion periods (July 2015 to March 2018). FINDINGS: Using interrupted time series analysis, an immediate significant change in HO-CDI was observed after intervention (P=0.03), although a downward trend was not observed over this period (P=0.19). Trends in PPED consumption significantly changed over this period (P=0.02). DOT of carbapenems decreased immediately after the intervention began (P<0.01). A Poisson regression analysis showed that PPED consumption and DOT of carbapenems were independent factors affecting HO-CDI (P=0.039 and 0.016, respectively). CONCLUSION: We revealed that DOT of carbapenems and use of PPED were associated with the HO-CDI ratio and that both interventions reduced the rate of HO-CDI. This is the first report on the impact of hospital ward cleaning with PPED on the reduction of HO-CDI.

Differing effects of liraglutide on gastric emptying in Japanese patients with type 2 diabetes.

This study was performed to clarify the influence of liraglutide on gastric emptying in Japanese patients with type 2 diabetes. In 16 patients, the [(13) C]-acetate breath test was performed to compare gastric emptying before and after liraglutide treatment. We found two patterns of response, with gastric emptying being delayed by liraglutide in seven patients (delayers) and not delayed in nine patients (non-delayers). The mean increase of the maximum gastric emptying time was 31 ± 4 min (p < 0.01 vs. baseline) in the delayers, while it was only 2 ± 3 min (p = 0.60 vs. baseline) in the non-delayers. The delayers showed a greater early decrease of AUC-PG from 0 to 60 min, despite no increase of the plasma insulin level compared with non-delayers. In conclusion, the effect of liraglutide treatment on gastric emptying shows heterogeneity, and patients can be classified as delayers or non-delayers.

Increased RP105-Negative B Cells in IgG4-Related Disease.

Four patients with IgG4-related disease (IgG4-RD) showed increased percentages of RP105-negative B cells in the peripheral blood. Case 1: A 66-year-old man having retroperitoneal fibrosis had 18.8% of RP105-negative B cells. Oral prednisolone improved the affected lesions and the percentage of RP105-negative B cells decreased (3.2%) after the treatment. Case 2: A 53-year-old man with retroperitoneal fibrosis had 27.9% of RP105-negative B cells. Case 3: A 38-year-old man with follicular hyperplasia showed increased percentage of RP105-negative B cells (8.3%). Case 4: A 60-year-old man with interstitial nephritis had 27.5% of RP105-negative B cells. The treatment decreased the numbers of RP105-negative B cells. Increased numbers of RP105-negatvie B cells is possibly associated with disease activity of IgG4-RD. Analysis of expression of RP105 on B cells may be helpful in evaluation of disease activity of IgG4-RD.

Preparation of polymer gel dosimeters based on less toxic monomers and gellan gum.

New polymer gel dosimeters consisting of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol monoethyl ether monomethacrylate (TGMEMA), polyethylene glycol 400 dimethacrylate (9G), tetrakis (hydroxymethyl) phosphonium chloride as an antioxidant, and gellan gum as a gel matrix were prepared. They were optically analyzed by measuring absorbance to evaluate a dose response. The absorbance of the polymer gel dosimeters that were exposed to (60)Co γ-rays increased with increasing dose. The dosimeters comprising HEMA and 9G showed a linear increase in absorbance in the dose range from 0 to 10 Gy. The dose response depended on the 9G concentration. For others comprising HEMA, 9G and TGMEMA, the absorbance of the polymer gel dosimeters drastically increased above a certain dose, and then leveled off up to 10 Gy. The optical variations in these polymer gel dosimeters were also induced by x-irradiation from Cyberknife radiotherapy equipment. Furthermore, the exposed region of the latter polymer gel dosimeter exhibited a thermo-responsive behavior.

Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

Cross talk between hedgehog and epithelial-mesenchymal transition pathways in gastric pit cells and in diffuse-type gastric cancers.

We previously reported hedgehog (Hh) signal activation in the mucus-secreting pit cell of the stomach and in diffuse-type gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is known to be involved in tumour malignancy. However, little is known about whether and how both signallings cooperatively act in diffuse-type GC. By microarray and reverse transcription-PCR, we investigated the expression of those Hh and EMT signalling molecules in pit cells and in diffuse-type GCs. How both signallings act cooperatively in those cells was also investigated by the treatment of an Hh-signal inhibitor and siRNAs of Hh and EMT transcriptional key regulator genes on a mouse primary culture and on human GC cell lines. Pit cells and diffuse-type GCs co-expressed many Hh and EMT signalling genes. Mesenchymal-related genes (WNT5A, CDH2, PDGFRB, EDNRA, ROBO1, ROR2, and MEF2C) were found to be activated by an EMT regulator, SIP1/ZFHX1B/ZEB2, which was a target of a primary transcriptional regulator GLI1 in Hh signal. Furthermore, we identified two cancer-specific Hh targets, ELK1 and MSX2, which have an essential role in GC cell growth. These findings suggest that the gastric pit cell exhibits mesenchymal-like gene expression, and that diffuse-type GC maintains expression through the Hh-EMT pathway. Our proposed extensive Hh-EMT signal pathway has the potential to an understanding of diffuse-type GC and to the development of new drugs.

Optoelectronic tweezers as a tool for parallel single-cell manipulation and stimulation.

Optoelectronic tweezers (OET) is a promising approach for the parallel manipulation of single cells for a variety of biological applications. By combining the manipulation capabilities of OET with other relevant biological techniques (such as cell lysis and electroporation), one can realize a true parallel, single-cell diagnostic and stimulation tool. Here, we demonstrate the utility of the OET device by integrating it onto single-chip systems capable of performing in-situ, electrode-based electroporation/lysis, individual cell, light-induced lysis, and light-induced electroporation.

Effects of ultrasound on the proliferation and differentiation of cementoblast lineage cells.

BACKGROUND: The purpose of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) stimulation on the proliferation and differentiation of cementoblast lineage cells. METHODS: An immortalized human periodontal ligament cell line (HPL) showing immature cementoblastic differentiation was used. Cultured HPL cells were subjected to LIPUS exposure (frequency = 1 MHz; pulsed 1:4; intensity = 30 mW/cm(2)) or sham exposure for 15 minutes per day. Expression levels of alkaline phosphatase (ALP), type I collagen (Col-I), runt-related gene 2 (Runx2), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OPN) mRNA were analyzed with real-time polymerase chain reaction analysis. Furthermore, ALP activity, collagen synthesis, and protein level of Runx2 were examined after 6 days of LIPUS exposure. RESULTS: mRNA and protein levels of ALP, Col-I, and Runx2 were significantly increased by LIPUS exposure compared to controls, whereas BSP, OCN, and OPN mRNA expression could not be detected in HPL cells, irrespective of LIPUS exposure. CONCLUSION: LIPUS enhanced ALP activity, collagen synthesis, and Runx2 expression of HPL cells, which provides important insight into the promotion of early cementoblastic differentiation of immature cementoblasts.

Adenosine A2A receptor antagonists: blockade of adenosinergic effects and T regulatory cells.

The intensity and duration of host responses are determined by protective mechanisms that control tissue injury by dampening down inflammation. Adenosine generation and consequent effects, mediated via A2A adenosine receptors (A2AR) on effector cells, play a critical role in the pathophysiological modulation of these responses in vivo. Adenosine is both released by hypoxic cells/tissues and is also generated from extracellular nucleotides by ecto-enzymes e.g. CD39 (ENTPD1) and CD73 that are expressed by the vasculature and immune cells, in particular by T regulatory cell. In general, these adenosinergic mechanisms minimize the extent of collateral damage to host tissues during the course of inflammatory reactions. However, induction of suppressive pathways might also cause escape of pathogens and permit dissemination. In addition, adenosinergic responses may inhibit immune responses while enhancing vascular angiogenic responses to malignant cells that promote tumor growth. Novel drugs that block A2AR-adenosinergic effects and/or adenosine generation have the potential to boost pathogen destruction and to selectively destroy malignant tissues. In the latter instance, future treatment modalities might include novel 'anti-adenosinergic' approaches that augment immune clearance of malignant cells and block permissive angiogenesis. This review addresses several possible pharmacological modalities to block adenosinergic pathways and speculates on their future application together with impacts on human disease.

Increase of RP105-lacking activated B cells in the peripheral blood and salivary glands in patients with Sjögren's syndrome.

OBJECTIVE: To quantify the activated B cells in the peripheral blood and salivary glands of patients with Sjögren's syndrome (SS) by analyzing the expression of RP105 molecule on the B cells. METHODS: The expression of RP105 on the peripheral blood B cells of patients with SS (19 cases) was analyzed by flow cytometry. RP105-positive and negative B cells were sorted and cultured in vitro and the amount of immunoglobulins (IgG and IgM) produced in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Salivary gland biopsy samples from 9 SS patients were histologically evaluated and the sequential frozen sections were separately immunostained by anti-RP105 and anti-CD20 monoclonal antibodies. RESULTS: A significantly higher proportion of peripheral blood RP105-negative B cells was found in SS patients than in healthy individuals. RP105-negative, but not positive, B cells from SS patients were capable of producing IgG and IgM spontaneously in vitro, which was enhanced by the addition of Staphylococcus aureus Cowan I strain (SAC) or IL-6. Salivary glands from 2 of 9 SS patients were found to have lymphoid follicles whose germinal centers consisted of RP105-negative B cells. Moreover, a larger proportion of B cells extensively infiltrating the area other than lymphoid follicles was also RP105-negative. CONCLUSION: RP105-negative B cells, a subset of highly activated and well differentiated B cells, which are increased in number in the peripheral blood and extensively infiltrate salivary glands, may be responsible for the production of class-switched immunoglobulin in SS. In addition, those cells might be associated with the inflammation and tissue damage of the salivary glands.

Hypoxia-dependent anti-inflammatory pathways in protection of cancerous tissues.

The evolutionarily selected tissue-protecting mechanisms are likely to be triggered by an event of universal significance for all surrounding cells. Such an event could be damage to blood vessels, which would result in local tissue hypoxia. It is now recognized that tissue hypoxia can initiate the tissue-protecting mechanism mediated by at least two different biochemical pathways. The central message of this review is that tumor cells are protected from immune damage in hypoxic and immunosuppressive tumor microenvironments due to the inactivation of anti-tumor T cells by the combined action of these two hypoxia-driven mechanisms. Firstly, tumor hypoxia-produced extracellular adenosine inhibits anti-tumor T cells via their G(s)-protein-coupled and cAMP-elevating A2A and A2B adenosine receptors (A2AR/A2BR). Levels of extracellular adenosine are increased in tumor microenvironments due to the changes in activities of enzymes involved in adenosine metabolism. Secondly, TCR-activated and/or tumor hypoxia-exposed anti-tumor T cells may be inhibited in tumor microenvironments by Hypoxia-inducible Factor 1alpha (HIF-1alpha) Hence, HIF-1alpha activity in T cells may contribute to the tumor-protecting immunosuppressive effects of tumor hypoxia. Here, we summarize the data that support the view that protection of hypoxic cancerous tissues from anti-tumor T cells is mediated by the same mechanism that protects normal tissues from the excessive collateral damage by overactive immune cells during acute inflammation.

The association of Behçet's disease with myelodysplastic syndrome in Japan: a review of the literature.

OBJECTIVE: [corrected] To determine the clinical characteristics of patients with myelodysplastic syndrome (MDS)-associated Behçet's disease (BD) in Japan. METHODS: 54 Japanese cases of MDS-associated BD obtained from the literature and from our own clinical experience were reviewed. The clinical features of MDS-associated BD were compared with those of the 1991 nationwide BD survey in Japan. RESULTS: In MDS-associated BD, the average age at onset was 42.6 years, which was 6.9 years later than for all BD patients; females developed disease more frequently than males (male: female ratio = 0.80). In MDS-associated BD cases, the occurrence of eye lesions was significantly lower, the frequency of intestinal lesions was markedly higher, and the rate of HLA-B51 positivity was lower than that in all BD. BD and MDS developed nearly simultaneously in 49.0% of cases; BD preceded MDS in 31.4% of the cases. The distribution of the age at BD onset showed two peaks, one in the 3rd decade and the other in the 6th decade. Females were more likely to develop younger-onset disease, while men were more likely to develop older-onset MDS-associated BD. Furthermore, in the older-onset group, BD was diagnosed together with or after the diagnosis of MDS, while half of the younger-onset group developed BD earlier than MDS. CONCLUSION: MDS-associated BD patients form a distinct subset of patients. There may, in fact, be two major groups of MDS-associated BD patients based on age, gender, and temporal relationship of the two diseases.

Prevention of steroid-induced osteonecrosis of femoral head in systemic lupus erythematosus by anti-coagulant.

Although osteonecrosis of femoral head (ONF) is one of the serious complications in systemic lupus erythematosus (SLE) associated with corticosteroid therapy, there has been few trials of prevention of ONF described. We aimed to prevent ONF in steroid-treated SLE patients using anticoagulant, warfarin, conducting a multicenter prospective study. Sixty newly diagnosed SLE patients requiring 40 mg/day or more prednisolone were alternately assigned to either of two groups; a warfarin group and a control one. Warfarin (1 to approximately 5 mg/day) was started together with the beginning of steroid therapy and continued at least for three months. Patients were observed for the development of silent ONF by magnetic resonance imaging (MRI) and symptomatic ONF by plain radiography for over five years. The warfarin group consisted of 31 patients (62 hips) and the control one 29 patients (58 hips). Silent ONF developed in 13 hips (21%) and 19 hips (33%) in the warfarin group and the control group, respectively (P = 0.13). On the other hand, warfarin tended to prevent symptomatic ONF; only three hips of 62 (4.8 %) in the warfarin group and eight hips of 58 (14%) in the control group (P = 0.08) developed silent ONF. It was also found that silent ONF developed, if it did, very early; within three months in 16 of 18 patients (89%). Among risk factors for silent ONF, steroid pulse therapy was most outstanding and it seemed to overcome the effect of warfarin. Taken together, for the time being, anti-coagulant therapy, if not significantly sufficient, may be of use for the prevention of steroid-induced ONF in SLE. We consider that this study added to important evidence for the pathogenesis and prevention of ONF.

Ex vivo CD(+) T-cell cytokine expression from patients with Sjögren's syndrome following in vitro stimulation to induce proliferation.

OBJECTIVE: To assess ex vivo CD4(+) T-cell cytokine expression from patients with primary Sjögren's syndrome (SS) following in vitro stimulation to induce proliferation, as proliferation is closely related to differentiation of cytokine-producing cells. METHODS: Peripheral blood mononuclear cells (PBMCs) separated from primary SS patients (n = 28) and controls (n = 25) were analysed. PBMCs were stimulated with concanavalin A followed by phorbol 12-myristate 13-acetate and ionomycin. Intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL)-4 in proliferating CD4(+) T cells were assessed by flow cytometry. The proportion of cytokine-producing cells and proliferating cells in each division cycle was assessed using [5(and 6)-carboxyfluorescein diacetate, succinimidyl ester]-labelled CD4(+/-) T cells. RESULTS: The proportion of IFN-gamma+ proliferating CD4(+) T cells in each cell division cycle from extraglandular SS was increased in glandular SS patients compared glandular SS patients with controls (P<0.05 approximately 0.01). The percentage of IFN-gamma single positive proliferating CD4(+) T cells was greater in extraglandular SS patients (26.7+/-14.1%) compared with glandular SS (9.9 +/- 9.1%) (P<0.01) and controls (9.4 +/- 5.8%) (P<0.001). There was no significant difference in the percentages of IL-4(+) proliferating CD4(+) T cells among the groups. However, the proliferating response of CD4(+) T cells was significantly decreased in extraglandular SS patients (percentage of proliferating cells 38.4 +/- 18.6%) compared with that in glandular SS patients (64.2 +/- 17.2%) (P<0.05) and controls (63.1+/-10.6%) (P<0.01). CONCLUSIONS: CD4(+) T cells from extraglandular SS patients may have a predisposition for entry into the IFN-gamma-producing effector pathway as a result of the stimulations. These results are helpful for understanding the immunological difference between glandular and extraglandular SS and the mechanisms of disease progression.

Very early development of steroid-associated osteonecrosis of femoral head in systemic lupus erythematosus: prospective study by MRI.

The objective of this study was to define prospectively the early development of corticosteroid-induced osteonecrosis of femoral head (ONF) in patients with systemic lupus erythematosus (SLE) and to identify the association of initial steroid treatment with the development of early (silent) ONE Forty-five patients who were newly diagnosed as having SLE and required 40 mg/day or more prednisolone were enrolled. To detect silent ONF, examinations using magnetic resonance imaging (MRI) were done three months after starting steroid therapy, followed by every year's MRI and plain radiography for over five years. Clinical and laboratory data were compared between silent ONF and non-ONF groups. Of 45 patients, 15 (33%) developed silent ONF and five (11%) symptomatic ONE It was of interest that MRI detected silent ONF very early (by three months) in 14 patients (93%). It should be noted that pulse therapy with 1000 mg/day methylprednisolone was found to be done very frequently (13 of 15, 87%) in the silent ONF group compared to non-ONF group (11 of 30, 37%) (P < 0.01) although other clinical features were not significantly different between both groups. High dose corticosteroids caused elevation of serum levels of total cholesterol, albumin, and leukocyte count in most of patients. The degree of elevation of those parameters at one or three months was more prominent in the silent ONF group. In particular, the change ratio of total cholesterol at one month was outstanding in the silent ONF group compared to non-ONF group (0.551 versus 0.374, P < 0.05). In conclusion, pathological ONF develops very early in one-third of SLE patients who received high dose corticosteroids and steroid pulse therapy could be a significant risk factor. An abrupt elevation of serum total cholesterol and/or sensitivity to steroids seem to be associated with the pathogenesis of ONF.

Impact of reproductive experience on women's smoking behaviour in Japanese nurses.

OBJECTIVES: The objectives of this study were to describe current and past smoking behaviour of female Japanese nurses, to examine factors associated with developing and stopping a smoking habit, and to examine how their reproductive experiences affect their smoking behaviour. STUDY DESIGN: A cross-sectional study. This study was a baseline survey of a prospective occupational cohort study. METHODS: A self-administered survey of 1748 female Japanese nurses aged over 20 years was conducted in Gunma prefecture, Japan, in 1999. RESULTS: Overall, 27.2% developed a smoking habit (current smokers, 19.8%; ex-smokers, 7.4%) and 72.2% were never smokers. Logistic regression analysis showed that the type of nursing certificate was statistically associated with developing a smoking habit. It also showed that the type of nursing certificate, work place, marital status and current pregnancy were statistically associated with smoking cessation. Currently pregnant women were more likely to stop smoking than non-pregnant women (adjusted odds ratio, 3.18; 95% confidence intervals, 1.25-8.06). For women aged 20-29 years, the proportions of current smokers, ex-smokers and never smokers among pregnant women were 11.5, 23.1 and 65.4%, respectively; among non-pregnant women of this age, the values were 22.3, 4.3 and 73.4%, respectively. There was a statistically significant difference in such proportions between the two groups (chi2=19.27; P<0.0001). More than half of the ex-smokers who were currently pregnant had stopped smoking in the last 12 months. Smoking behaviour showed no statistically significant difference between women who had had at least one delivery and women who had not. CONCLUSIONS: The results suggest that pregnancy provides a good opportunity for smoking cessation, but a large proportion of women who successfully quit smoking during pregnancy relapse after delivery.

Increased entry of CD4+ T cells into the Th1 cytokine effector pathway during T-cell division following stimulation in Behcet's disease.

OBJECTIVES: To investigate the relationship between the production of Th1/Th2 cytokines and cell kinetics, cell division and proliferation in patients with Behçet's disease (BD). METHODS: Peripheral venous blood was drawn from patients with BD (n = 24; 10 patients with active and 14 patients with inactive BD) and normal subjects (n = 22). Peripheral blood mononuclear cells were separated immediately and were cultured with concanavalin A (Con A) followed by phorbol 12-myristate 13-acetate and ionomycin (PMA+Ion). Intracellular cytokine production of interferon-gamma (IFN-gamma) (Th1) and IL-4 (Th2) in CD4(+) T cells was determined by flow cytometry. Furthermore, CD4(+) T cells labelled with CFSE [5 (and 6) carboxyfluorescein diacretate, succinimidyl ester] were stimulated and the cells were analysed for entry into the cytokine production effector pathway during cell division in active BD and normal subjects. RESULTS: In active BD, enhanced entry into the Th1 response effector pathway of CD4(+) T cells was observed after stimulation with Con A followed by PMA+Ion. Analysis of CD4(+) T cells at an identical cell division number in response to Con A followed by PMA+Ion revealed that IFN-gamma-producing cells were increased in active BD patients compared with normal subjects. These results suggest that the Th1 response of dividing CD4(+) T cells is predominantly operating in active BD. Dividing CD4(+) T cells stimulated with Con A followed by PMA+Ion showed a phenotype of activated effector memory T cells (CD45RA(low), CD45RO(+), CD69(high)). CONCLUSIONS: Cell kinetics play a crucial role in Th1 cell differentiation and pathophysiology in BD.

Retinal findings in systemic sclerosis: a comparison with nailfold capillaroscopic patterns.

OBJECTIVE: To determine the prevalence of retinal disease in systemic sclerosis (SSc) and to characterise the findings of retinopathy. Additionally, to analyse the association of retinal disease with other clinical/laboratory findings, particularly the findings of nailfold capillaries in patients with SSc. METHODS: Photographs of the ocular fundi were taken and were evaluated by an ophthalmologist who was unaware of the SSc status of the patients. The nailfold capillaries were analysed with a dermatoscope. Patients were divided into two groups according to the presence (group A) or absence (group B) of retinal disease. RESULTS: Retinal findings of the patients with SSc consisted of hard exudates, vascular tortuosity, microhaemorrhage, and macular degeneration. The prevalence of retinal disease among the patients with SSc was 34% (10/29), compared with 8%(3/38) among the controls (p=0.011). The mean systolic blood pressure and the age of the patients in group A were significantly higher than those in group B. However, there was no significant difference in the nailfold capillary damage between groups A and B. CONCLUSION: Retinal abnormalities are often seen in patients with SSc and they may reflect the vascular changes characteristic of SSc. However, retinal changes may differ in quality from the changes of nailfold capillaries.